Detection of the nondefoliating pathotype of Verticillium dahliae in infected olive plants by nested PCR

11 páginas, 4 figurasAn increasing incidence and distribution of verticillium wilt has occurred in the last few years in newly established olive orchards in southern Spain. This spread of the disease may result from use of Verticillium dahliae-infected planting material. The early in planta detection of the pathogen would aid the implementation of certification schemes for pathogen-free planting material. In this work, a nested polymerase chain reaction (PCR) method was developed for the in planta detection of the nondefoliating (ND) V. dahliae pathotype, aimed especially at nurseryproduced olive plants. For this purpose, specific primers were designed from the sequence of a 1958-bp random amplified polymorphic DNA (RAPD) marker of ND V. dahliae, and a procedure for the extraction of PCR-quality total genomic DNA from infected root and stem tissues of young olive plants was tested and further optimized. Nested PCR assays detected ND V. dahliae in 4- to 14-month-old artificially infected plants of three olive cultivars. The ND-specific PCR product was not amplified from total genomic DNA extracted from olive plants infected with the defoliating V. dahliae pathotype. Detection of the ND pathotype was effective from the very earliest moments following artificial inoculation of olive plants with a V. dahliae conidial suspension. Also, detection was achieved in inoculated, though symptomless, olive plants as well as in plants that were symptomatic but became symptomless by 217 days after inoculation.Comisión Interministerial de Ciencia y Tecnología (CICYT), España. 1FD97-0763-C03-01; Comunidad Europea, contrato no. QLK5-CT1999-01523.Peer reviewe